Aberrant androgen synthesis causes disease and compromises the health of women, but therapies targeting the enzyme responsible, 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), have been difficult to develop due to an incomplete understanding of the regulation of the 17,20-lyase activity of this protein. A more complete understanding requires the development of novel paradigms and the application of new methods that are proposed in this application. We hypothesize that the 17,20-lyase activity of P450c17, directly responsible for androgen synthesis, is regulated at the molecular level by redox potential and at a macro-molecular level by protein-protein interactions. The specific aims of this proposal are: 1) To develop cyclic voltammetry (CV) to record directly the redox potential of P450c17, enabling the electrochemical interrogation of the activity of the enzyme and the molecular regulation of activity, and 2) To develop fluorescence resonance energy transfer (FRET) techniques to provide direct information relating to protein-protein interactions influencing P450c17 activity and androgen synthesis in living cells. CV will be used to measure redox potentials of purified recombinant human, bovine, porcine and mutant human P450c17 proteins in the presence or absence of substrates, the redox partner protein NADPH-cytochrome P450 reductase (CPR) and the allosteric mediator cytochrome b5. Catalytic currents will be also monitored with oxygen introduction. FRET will be monitored using fusion constructs of P450c17, CPR and cytochrome b5 with fluorescent proteins (eCFP and eYFP). These will be expressed transiently in human adreno-cortical cells (H295) at various molar ratios before and after forskolin and cAMP stimulation. Both techniques can examine events in real time and will complement one another by examining the electronic consequences of protein-protein interactions (CV), documenting interactions in live cells and determining what promotes them (FRET). These techniques, and the paradigm they will establish, have immediate relevance to androgen synthesis, but equally have application in the investigation of a variety of phenomena involving interactions among P450 enzymes in metabolic cascades.